Since the production of tissue plasminogen activator (tPa) as the first marketed recombinant therapeutic drug in 1987, the methods for producing recombinant proteins have remained largely unchanged. For example, Chinese Hamster Ovary (CHO) cells have been the cell line of choice for creating stable recombinant polypeptide production cell lines. CHO cells, however, like other cancer-derived cell lines, are inherently unstable because of their aneuploidy and predilection for chromosomal rearrangements making the process of finding stable clones difficult. Although relative production increases in the last 25 years have been substantial from 50-100 mg/l to 1-5 g/l, the yield increases can be attributed to process improvements rather than improvements on the technology used (see, e.g., the review by De Jesus & Wurm, European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 184-188). Thus, current methodology for producing recombinant proteins relies on the creation of a clonal cell line that stably produces protein at high expression levels. This process is labor intensive and can take a year or longer for creation of a suitable production-based cell line for production.
Alternatively, transient gene expression may be used to achieve a transient moderate yield of a recombinant polypeptide (e.g., reviewed by Hopkins et al., Methods Mol Biol, 2012, 801:251-68). Although this methodology is simpler and more rapid (e.g., with the possibility to produce at a gram per liter in a two-week period), the possibility to scale this technology beyond 100 liters does not yet exist. Thus, there is a need for a scalable system for generating stable recombinant polypeptide producing cells in a more rapid and cost effective manner.
Multi-component therapies based on recombinant polyclonal proteins represent promising new drugs for the treatment of various diseases and disorders including infectious diseases, cancer, neurological disorders, inflammation and immune disorders, and cardiovascular diseases. Polyclonal proteins, such as vaccines and polyclonal antibodies, are uniquely suited to treat pathogens and other disease causing agents because they simultaneously target multiple epitopes and, therefore, decrease the selective pressure for the development of resistant strains. For example, the effectiveness of hyperimmune immunoglobulins is rarely affected by mutations in the target pathogen, because these immunoglobulins consist of complex pools of antibodies derived from individuals with high titers against a specific infectious disease. Exemplary hyperimmune immunoglobulins include CytoGAM® (which targets cytomegalovirus), VZIG (which targets varicella virus), HBIG (which targets hepatitis B virus), and RIG (which targets rabies virus). Despite their effectiveness, however, hyperimmune immunoglobulins are subject to batch-to-batch variability, limited availability, and carry the risk of blood-borne disease transmission. In addition, relatively high doses are needed because only a small percentage of the component antibodies recognize epitopes in the agent of interest.
Monoclonal antibody therapies have also been developed; however, the use of monoclonal antibodies for the treatment of infectious disease has so far been largely unsuccessful. Currently, the only monoclonal antibody on the market against an infectious disease agent is Synagis (palivizumab), which targets Respiratory Syncytial Virus (RSV). Although Synagis has successfully prevented infection in premature infants, its efficacy in other applications, such as prevention of disease in children with cystic fibrosis or as a treatment in adults with stem cell transplants has been unclear. In other studies, some monoclonal antibody therapies are able to provide a degree of protection in animal models of infectious disease, but the required doses are often high. Further, a high degree of genetic diversity and variable expression among bacterial strains reduces the likelihood that a single monoclonal treatment will be effective against all strains.
Although immunoglobulins (e.g., passive immunotherapy) have been employed in human medicine since the late 1800s and monoclonal antibodies since the 1980s, human recombinant polyclonal antibodies (HRPAs) are a relatively new form of immunotherapy. Like intravenous immunoglobulins (IVIGs), HRPAs are able to overcome multiple mutational challenges without significant loss of efficacy, thereby reducing selective pressure that leads to the emergence of resistant strains. However, unlike serum derived polyclonal immunoglobulins, the recombinant nature of HRPA therapy avoids the variability in composition and availability, as well as the risk of blood borne disease transmission inherent in IVIG preparations. In addition, synergistic activity of a large group of highly specific antibodies within an HRPA may enhance passive immunotherapy efficacy far beyond what can be accomplished with monoclonals or IVIGs.
A number of methods for the manufacture of recombinant antibodies have been described previously to generate large amounts of recombinant protein for clinical uses. Many of these methods rely on transfection of plasmid DNA followed by random integration of the gene into the host cell genomic DNA, selection of individual progenitor clones with high expression levels, and expansion to generate master cell lines for production. Random integration into the genome can result in positional effects due to insertion in “non-permissive” regions of the genome. Once highly expressing clones are obtained, expression may be unstable, decreasing over time due to gene silencing mechanisms such as methylation or heterochromatin formation. Bulk transformed cell populations are also used, but only for selecting specific highly stable producer cell lines. The number of methods for production of recombinant polyclonal proteins is more limited. Moreover, it is well-understood in the field of recombinant protein expression that a mixture of cell lines is unstable (see, e.g., Migliaccio et al., Gene 256 (2000) 197-214, which describes that integration of extraneous DNA into a cell's genome is often inherently unstable and Nielsen et al., Mol Biotechnol (2010) 45:257-266, which discusses instability and bias of antibody producing cells in mixed cell lines).
To circumvent the positional effects observed with random integration techniques, methods for site-specific integration of expression constructs into the genome have been described (see, e.g., U.S. Pat. Nos. 4,959,317 and 5,654,182; WO 98/41645; WO 01/07572; WO 2005/042774 and WO 2004/061104). For example, patent application WO 2004/061104 describes a method for the production of recombinant polyclonal proteins, including antibodies, using a library of vectors in such a way that relative expression levels of these vectors are maintained over time. This method makes use of a site-specific Flp recombinase to ensure that integration of the recombinant protein expression cassette occurs at FRT site(s) present in the genome that is presumed to be permissive for gene expression. However, the use of site-specific Flp recombinase for the production of polyclonal antibodies can result in low expression levels during production, loss of expression, and genome instability similar to that observed with randomly integrated DNA because (a) FRT site(s) have to be engineered into the genome of the expression cell line. To address this potential for instability and low inconsistent expression, a cell line can be selected for with the FRT site located in a particularly permissive site that confers high stability and expression (Zhou et al., J Biotech 147 (2010) 122-129).
As such, there remains an ongoing need for improved recombinant polyclonal proteins that are useful as therapeutic agents, and methods for producing stable populations of recombinant polyclonal proteins.